RNA interference (RNAi) mediated by short-interfering RNA (siRNA) is an emerging tool in basic science and is poised to offer new therapeutic modalities to treat various diseases. RNAi is an evolutionarily conserved, sequence specific mechanism triggered by double stranded RNA (dsRNA) that induces degradation of complementary target single stranded mRNA and “silencing” of the corresponding translated sequences (McManus and Sharp, Nature Rev. Genet., 3:737 (2002)). RNAi functions by enzymatic cleavage of longer dsRNA strands into biologically active siRNA sequences of about 21-23 nucleotides in length (Elbashir, et al., Genes Dev., 15:188 (2001)).
Inhibitory RNAs can be used downregulate or silence the translation of a gene product of interest. For example, it can be desirable to downregulate genes associated with various diseases and disorders. Although siRNAs have surpassed expectation when used to alter gene expression in the laboratory setting, RNAi-based therapies for the clinic are still limited by the availability of efficient delivery systems (Novina and Sharp, Nature, 430:161-163 (2004); Garber, Natl. Cancer Inst., 95(7):500-2 (2003)). An effective and safe nucleic acid delivery system is required for siRNA to be therapeutically useful.
Inhibitory RNAs that are administered “naked” to most subjects can be degraded by endogenous nucleases; and may not be able to cross cell membranes to contact and silence their target gene sequences. Viral vectors are relatively efficient gene delivery systems, but suffer from a variety of safety concerns, such as potential for undesired immune responses. Furthermore, viral systems are rapidly cleared from the circulation, limiting transfection to “first-pass” organs such as the lungs, liver, and spleen. In addition, these systems induce immune responses that compromise delivery with subsequent injections. As a result, nonviral gene delivery systems are receiving increasing attention (Worgall, et al., Human Gene Therapy, 8:37 (1997); Peeters, et al., Human Gene Therapy, 7:1693 (1996); Yei, et al., Gene Therapy, 1:192 (1994); Hope, et al., Molecular Membrane Biology, 15:1 (1998)).
Plasmid DNA-cationic liposome complexes are currently the most commonly employed nonviral gene delivery vehicles (Felgner, Scientific American, 276:102 (1997); Chonn, et al., Current Opinion in Biotechnology, 6:698 (1995)). Cationic liposome complexes, however, are large, poorly defined systems that are not suited for systemic applications and can elicit considerable toxic side effects (Harrison, et al., Biotechniques, 19:816 (1995); Li, et al., The Gene, 4:891 (1997); Tam, et al, Gene Ther., 7:1867 (2000)). As large, positively charged aggregates, lipoplexes are rapidly cleared when administered in vivo, with highest expression levels observed in first-pass organs, particularly the lungs (Huang, et al., Nature Biotechnology, 15:620 (1997); Templeton, et al., Nature Biotechnology, 15:647 (1997); Hofland, et al., Pharmaceutical Research, 14:742 (1997)). Although RNA lipoplexes are easily formulated and have been used successfully in a few applications, lipoplexes suffer from toxicity, especially to the vulnerable mucosal epithelium, and do not offer the potential for controlled or sustained release. Other liposomal delivery systems include, for example, the use of reverse micelles, anionic and polymer liposomes as disclosed in, e.g., U.S. Pat. No. 6,429,200; U.S. Patent Application No. 2003/0026831; and U.S. Patent Application Nos. 2002/0081736 and 2003/0082103, respectively.
Intravaginal delivery of inhibitory RNAs, including siRNA, is especially challenging but desirable because of the susceptibility of reproductive tissues to infectious diseases, which are potentially treatable by inhibitory RNA approaches (Valenta, Adv. Drug Deliver. Rev., 57:1692-1712 (2005)). To date, vaginal instillation of siRNA using commercial liposomes in murine models has led to silencing of endogenous genes (Zhang, et al., Mol. Ther., 14(3):336-42 (2006)) in the genital tract and protected against challenge from herpes simplex virus (Palliser, et al., Nature, 439(7072):89-94 (2006)).
It is therefore an object of the invention to provide compositions that can deliver high concentrations of inhibitory RNAs to cells and tissues.
It is another object of the invention to provide compositions that can deliver inhibitory RNAs to cells and tissues in a controlled and sustained manner over a period of at least one week.
It is yet another object of the invention to provide compositions that can penetrate deep into tissues and deliver inhibitory RNAs intracellularly.
It is still a further object of the invention to provide methods for making and using these inhibitory RNA delivery compositions.